Planta
Planta (1990)181:529-537
9 Springer-Verlag 1990
Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos Ron Kooijman*, Piet de Wildt, Wies van den Briel, Shu-hui Tan, Alan Musgrave**, and Herman van den Ende Department of Molecular Cell Biology, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, The Netherlands Abstract. Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic A M P (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated ceils. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella. Key words: Cyclic A M P - Cell recognition - Chlamydomonas (gametes) - Mating responses - Plant cell signalling - Wheat germ agglutinin
Introduction Sexual reproduction in the green alga Chlamydomonas eugametos is initiated when the flagella of mating-typeplus (mt +) gametes bind to those o f mating-type-minus (rot-) gametes, Large aggregates of cells are formed (agglutination) in which cells sort themselves out into pairs * Present address: Wilhelmina Kinderziekenhuis, Nieuwegracht
137, NL-3512 LK Utrecht, The Netherlands ** To whom correspondence should be addressed Abbreviations: db-cAMP=dibutyryl-cAMP; FTA=flagellar tip activation; Mab=monoclonal antibody; mt-/mt§ minus/plus; WGA = wheat-germ agglutinin
that eventually fuse. Fusion is preceded by several agglutination-induced reactions in the flagella and cell body: (1) flagellar beating changes from a swimming stroke into a twitching movement (Homan et al. 1980); (2) flagellar-tip activation (FTA) occurs, which is the rounding of the flagellar tip as a consequence of the elongation of some outer microtubules (Mesland et al. 1980; Elzenga et al. 1982; Crabbendam et al. 1984); (3) there is a transient increase in agglutinability (Demets et al. 1988); (4) there is transport of agglutinins to the flagellar tip (tipping; Homan et al. 1987); (5) local cell-wall lysis occurs between the bases of the flagella and a plasma papilla protrudes through the anterior cell wall, allowing the gamete to fuse with its partner. The dynamics of these mating responses and their role in mating have been described by Musgrave and van den Ende (1987). The flagellar components which are responsible for adhesion have been identified and characterized for C. eugametos (Musgrave et al. 1981; H o m a n et al. 1982; Klis et al. 1985; Crabbendam et al. 1987), C. reinhardtii (Collin-Osdoby and Adair 1985; Adair et al. 1982) and C. moewusii (Samson et al. 1987). In C. eugametos, these so-called agglutinins are long stringy, high-molecularweight glycoproteins extrinsically attached to the flagellar membrane (Musgrave etal. 1981; H o m a n etal. 1982). Previously, we presented several arguments for a unipolar binding model in which the m t - agglutinins bind to the mt + agglutinins, induce the formation of an intracellular signal and consequently trigger mating responses in both gametes (Homan et al. 1988; Kooijman et al. 1989). While the agglutinins and mating responses in all the species are well characterised, the signal transduction mechanism is only recently becoming elucidated. Calcium has often been invoked as a signal in mating C. reinhardtii gametes. Kaska et al. (1985) and Bloodgood and Levin (1983) reported the release of immobilized intracellular Ca z + during agglutination and Snell et al. (1982) reported the inhibition of cell fusion by lidocaine. However, intracellular Ca z+ concentrations have never been measured and, apart from the isolated report by Claes (1980), no one has been able
530 artificially to raise the C a 2 + level by using c a l c i u m i o n o p h o r e s such as A23187 a n d i n d u c e sexual responses (Pasquale a n d G o o d e n o u g h 1987; see also this report). In contrast, a role for cyclic A M P ( c A M P ) in C. eugametos gametes was i m p l i c a t e d by Pijst et al. (1984), who observed a t r a n s i e n t , b u t d r a m a t i c increase in the c A M P level as a n i m m e d i a t e c o n s e q u e n c e o f a g g l u t i n a t i o n , a n d a s t i m u l a t i n g effect o f d i b u t y r y l - c A M P ( d b - c A M P ) o n the fusion c o m p e t e n c e o f m a t i n g cells (Pijst 1985). Pasquale a n d G o o d e n o u g h (1987) developed this theme further a n d d e m o n s t r a t e d t h a t in C. reinhardtii, a g g l u t i n a t i o n also triggers a n increase in the c A M P level. They d e m o n s t r a t e d t h a t c A M P is i n v o l v e d in sexual signalling by s h o w i n g t h a t d b - c A M P c a n i n d u c e all m a t i n g responses in n o n - m a t i n g gametes. This h a d n o t been possible in C. eugametos. In reassessing the role o f c A M P in this species, we studied the effect of w h e a t - g e r m agglut i n i n ( W G A ) o n c A M P levels in gametes because this lectin c a n evoke all the m a t i n g responses, a n d we tested the effect o f e x o g e n e o u s c A M P o n n o n - m a t i n g gametes in the light a n d in darkness. O n l y those in d a r k n e s s r e s p o n d e d . I n a d d i t i o n , the presence o f a d e n y l a t e cyclase in C. eugametos was tested to c o m p l e m e n t the earlier s t u d y o n p h o s p h o d i e s t e r a s e a n d c A M P - d e p e n d e n t prot e i n - k i n a s e activity in this alga (Pijst 1985).
Material and methods Cell cultures. The rot- strain 5.39.4 and the mt + strain 17.17.2 of Chlamydomonas eugametos were obtained as described by Schuring et al. (1987) and the cells were cultivated on an agar medium in a 12 h dark/12 h light regimen (Mesland 1976). Gamete suspensions were obtained by flooding two- to four-week-old cultures with a 10mM Hepes [4-(2-hydroxyethyl]-l-piperazineethanesulphonic acid]) buffer, pH 7.6. Non-illuminated cells were obtained by placing the cells in darkness immediately after flooding. Illumination of gametes occurred at a photon fluence rate of 34 Ixmolm - 2. s - 1 using white light from fluorescent lamps. The adenylatecyclase activity was assessed in the mt+ strain 17.17.2. For the other experiments, the mt- strain 5.39.4 was used.
R. Kooijman et al. : cAMP signal in Chlamydomonas gametes exposed papillar membrane and were observed as a fluorescent point between the flagellar bases. To determine the effect of db-cAMP on the flagellar agglutinability, control cells and db-cAMP-treated cells were fixed in 1.25% glutaraldehyde for 30 min. Subsequently, the fixative was removed by three washing steps in distilled water. The agglutinability of the fixed cells was quantified by determining the highest dilution of the suspension that still exhibited agglutination activity when mixed with live gametes of the opposite mating type. The fusion competence of the mt- cells was determined by mixing them with a tenfold excess of mt § cells. After 60 min, the vis-fi-vis pairs were fixed in 1.25% glutaraldehyde and the percentage of fusion-competent mt- cells was determined by the equation : No. vis-fi-vis pairs x 100%/No. mt- cells.
Determination of cAMP. For cAMP measurements, 100 ~1 aliquots (containing 1.106-2.106 cells) were extracted in 3 M perchloric acid as described by Pijst et al. (1984). The amount of cAMP was determined using a competition radioimmunoassay based on the method of Steiner et al. (1972), For this assay, a cAMP-specific antiserum together with [125I]cAMP tyrosine methyl ester as a competing agent (Amersham International, Amersham, UK), were used. Acetylation of cAMP, using acetic anhydride and triethylamine, resulted in a sensitivity of 2 fmol cAMP per tube. By incubating the samples with phosphodiesterase, it was shown that the measured values represented cAMP. Localization ofadenylate-cyclase activity. The flagella were amputated by pH-shock (Whitman et al. 1972) and separated from the cell bodies as described before (Kooijman et al. 1986). The flagellar membranes were fragmented by sonication and the cell bodies were homogenized by vortexing them with glass beads (0.5 mm diameter) as described by Pijst et al. (1984). Whole-cell homogenates, membrane fractions and cytoplasm fractions were obtained as described by Pijst et al. (1984). Adenylate-cyclase activities were determined according to Janssens et al. (1987).
Results Responses o f c A M P induced by W G A . The t e t r a v a l e n t lectin W G A can i n d u c e all the m a t i n g responses in C. eugametos gametes ( K o o i j m a n et al. 1989). T h a t c A M P
30
Reagents. Phorbol myristate acetate, db-cAMP and db-cGMP were obtained from Sigma, St. Louis, USA. Caffeine was purchased from BDH chemicals, Poole, UK. Calcium ionophore A23187 was purchased from Boehringer, Mannheim, FRG and WGA was obtained from Serva, Heidelberg, FRG. Mating reactions. Twitching was assessed by microscopical observation. Flagellar-tip activation (FTA) was observed and quantitated as described previously (Kooijman et al. 1986). The presence and localization of active agglutinins was assessed with the aid of the monoclonal antibody Mab 66.3 using an indirect immunofluorescence test (Kooijman et al. 1989). Monoclonal antibody Mab 66.3 binds to the binding site of the mt- agglutinin which is exposed on the active but not on the inactive form of the molecule (Homan et al. 1988; Kooijman et al. 1988). Cells were tested for local cell-wall lysis by enclosing a suspension of gametes on a microscope slide under a coverslip. After 5-10 rain, the preparation had dried out to the extent that the cell contents were squeezed out, resulting in the extrusion of a "balloon" between the bases of the flagella (F. Schuring, University of Amsterdam, The Netherlands, personal communication). Mating-structure activation was observed by indirect immunofluorescence assay using WGA at a concentration of 500 pg.ml- 1 or Mab 66.3. Wheat-germ agglutinin and Mab 66.3 bind to the
09 O
,r-
O-
0
i 2
9
i 4
.
,
i
9
9
6
i 8
9
i 10
2
time (min) Fig. l. Agglutination- and WGA-induced changes in the cAMP levels in Chlamydomonas gametes incubated in the light. CyclicAMP concentrations were determined after mixing mt+ and rotgametes (open symbols) or during the incubation of rot- gametes in 500 I.tg-ml-1 WGA (closed symbols). The cAMP levels measured during agglutination are the mean levels in the rot- and the mt+ cells
R. Kooijman et al. : c A M P signal in Chlamydomonas gametes 6O
531 Table 1. Mating responses of Chlamydomonas induced by W G A . Cells that were used in the experiment of Fig. 5 were tested for the induction of mating reactions using W G A in darkness and in light. After 1 min incubation with W G A , the cells were tested for the presence ( - - ) or absence ( - ) of twitching activity; FTA, tipping and cell-wall lysis were tested and quantified after a 30-min incubation period
A
4O 30-
Mating reaction
WGA (gg .ml- 1)
Dark
Light
Twitching Twitching Tipping FTA Cell-wall lysis Cell-wall lysis
25 50 50 50 50 500
-+ 24% 46% 0% 0%
+ + 36% 32% 0% 30%
20-
01
CO
100
9
9
0
'0
I
I
2
4
-6 E
I
9
6
I
8
I0
1
time (min)
d.
Planta (1990)181:529-537
9 Springer-Verlag 1990
Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos Ron Kooijman*, Piet de Wildt, Wies van den Briel, Shu-hui Tan, Alan Musgrave**, and Herman van den Ende Department of Molecular Cell Biology, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, The Netherlands Abstract. Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic A M P (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated ceils. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella. Key words: Cyclic A M P - Cell recognition - Chlamydomonas (gametes) - Mating responses - Plant cell signalling - Wheat germ agglutinin
Introduction Sexual reproduction in the green alga Chlamydomonas eugametos is initiated when the flagella of mating-typeplus (mt +) gametes bind to those o f mating-type-minus (rot-) gametes, Large aggregates of cells are formed (agglutination) in which cells sort themselves out into pairs * Present address: Wilhelmina Kinderziekenhuis, Nieuwegracht
137, NL-3512 LK Utrecht, The Netherlands ** To whom correspondence should be addressed Abbreviations: db-cAMP=dibutyryl-cAMP; FTA=flagellar tip activation; Mab=monoclonal antibody; mt-/mt§ minus/plus; WGA = wheat-germ agglutinin
that eventually fuse. Fusion is preceded by several agglutination-induced reactions in the flagella and cell body: (1) flagellar beating changes from a swimming stroke into a twitching movement (Homan et al. 1980); (2) flagellar-tip activation (FTA) occurs, which is the rounding of the flagellar tip as a consequence of the elongation of some outer microtubules (Mesland et al. 1980; Elzenga et al. 1982; Crabbendam et al. 1984); (3) there is a transient increase in agglutinability (Demets et al. 1988); (4) there is transport of agglutinins to the flagellar tip (tipping; Homan et al. 1987); (5) local cell-wall lysis occurs between the bases of the flagella and a plasma papilla protrudes through the anterior cell wall, allowing the gamete to fuse with its partner. The dynamics of these mating responses and their role in mating have been described by Musgrave and van den Ende (1987). The flagellar components which are responsible for adhesion have been identified and characterized for C. eugametos (Musgrave et al. 1981; H o m a n et al. 1982; Klis et al. 1985; Crabbendam et al. 1987), C. reinhardtii (Collin-Osdoby and Adair 1985; Adair et al. 1982) and C. moewusii (Samson et al. 1987). In C. eugametos, these so-called agglutinins are long stringy, high-molecularweight glycoproteins extrinsically attached to the flagellar membrane (Musgrave etal. 1981; H o m a n etal. 1982). Previously, we presented several arguments for a unipolar binding model in which the m t - agglutinins bind to the mt + agglutinins, induce the formation of an intracellular signal and consequently trigger mating responses in both gametes (Homan et al. 1988; Kooijman et al. 1989). While the agglutinins and mating responses in all the species are well characterised, the signal transduction mechanism is only recently becoming elucidated. Calcium has often been invoked as a signal in mating C. reinhardtii gametes. Kaska et al. (1985) and Bloodgood and Levin (1983) reported the release of immobilized intracellular Ca z + during agglutination and Snell et al. (1982) reported the inhibition of cell fusion by lidocaine. However, intracellular Ca z+ concentrations have never been measured and, apart from the isolated report by Claes (1980), no one has been able
530 artificially to raise the C a 2 + level by using c a l c i u m i o n o p h o r e s such as A23187 a n d i n d u c e sexual responses (Pasquale a n d G o o d e n o u g h 1987; see also this report). In contrast, a role for cyclic A M P ( c A M P ) in C. eugametos gametes was i m p l i c a t e d by Pijst et al. (1984), who observed a t r a n s i e n t , b u t d r a m a t i c increase in the c A M P level as a n i m m e d i a t e c o n s e q u e n c e o f a g g l u t i n a t i o n , a n d a s t i m u l a t i n g effect o f d i b u t y r y l - c A M P ( d b - c A M P ) o n the fusion c o m p e t e n c e o f m a t i n g cells (Pijst 1985). Pasquale a n d G o o d e n o u g h (1987) developed this theme further a n d d e m o n s t r a t e d t h a t in C. reinhardtii, a g g l u t i n a t i o n also triggers a n increase in the c A M P level. They d e m o n s t r a t e d t h a t c A M P is i n v o l v e d in sexual signalling by s h o w i n g t h a t d b - c A M P c a n i n d u c e all m a t i n g responses in n o n - m a t i n g gametes. This h a d n o t been possible in C. eugametos. In reassessing the role o f c A M P in this species, we studied the effect of w h e a t - g e r m agglut i n i n ( W G A ) o n c A M P levels in gametes because this lectin c a n evoke all the m a t i n g responses, a n d we tested the effect o f e x o g e n e o u s c A M P o n n o n - m a t i n g gametes in the light a n d in darkness. O n l y those in d a r k n e s s r e s p o n d e d . I n a d d i t i o n , the presence o f a d e n y l a t e cyclase in C. eugametos was tested to c o m p l e m e n t the earlier s t u d y o n p h o s p h o d i e s t e r a s e a n d c A M P - d e p e n d e n t prot e i n - k i n a s e activity in this alga (Pijst 1985).
Material and methods Cell cultures. The rot- strain 5.39.4 and the mt + strain 17.17.2 of Chlamydomonas eugametos were obtained as described by Schuring et al. (1987) and the cells were cultivated on an agar medium in a 12 h dark/12 h light regimen (Mesland 1976). Gamete suspensions were obtained by flooding two- to four-week-old cultures with a 10mM Hepes [4-(2-hydroxyethyl]-l-piperazineethanesulphonic acid]) buffer, pH 7.6. Non-illuminated cells were obtained by placing the cells in darkness immediately after flooding. Illumination of gametes occurred at a photon fluence rate of 34 Ixmolm - 2. s - 1 using white light from fluorescent lamps. The adenylatecyclase activity was assessed in the mt+ strain 17.17.2. For the other experiments, the mt- strain 5.39.4 was used.
R. Kooijman et al. : cAMP signal in Chlamydomonas gametes exposed papillar membrane and were observed as a fluorescent point between the flagellar bases. To determine the effect of db-cAMP on the flagellar agglutinability, control cells and db-cAMP-treated cells were fixed in 1.25% glutaraldehyde for 30 min. Subsequently, the fixative was removed by three washing steps in distilled water. The agglutinability of the fixed cells was quantified by determining the highest dilution of the suspension that still exhibited agglutination activity when mixed with live gametes of the opposite mating type. The fusion competence of the mt- cells was determined by mixing them with a tenfold excess of mt § cells. After 60 min, the vis-fi-vis pairs were fixed in 1.25% glutaraldehyde and the percentage of fusion-competent mt- cells was determined by the equation : No. vis-fi-vis pairs x 100%/No. mt- cells.
Determination of cAMP. For cAMP measurements, 100 ~1 aliquots (containing 1.106-2.106 cells) were extracted in 3 M perchloric acid as described by Pijst et al. (1984). The amount of cAMP was determined using a competition radioimmunoassay based on the method of Steiner et al. (1972), For this assay, a cAMP-specific antiserum together with [125I]cAMP tyrosine methyl ester as a competing agent (Amersham International, Amersham, UK), were used. Acetylation of cAMP, using acetic anhydride and triethylamine, resulted in a sensitivity of 2 fmol cAMP per tube. By incubating the samples with phosphodiesterase, it was shown that the measured values represented cAMP. Localization ofadenylate-cyclase activity. The flagella were amputated by pH-shock (Whitman et al. 1972) and separated from the cell bodies as described before (Kooijman et al. 1986). The flagellar membranes were fragmented by sonication and the cell bodies were homogenized by vortexing them with glass beads (0.5 mm diameter) as described by Pijst et al. (1984). Whole-cell homogenates, membrane fractions and cytoplasm fractions were obtained as described by Pijst et al. (1984). Adenylate-cyclase activities were determined according to Janssens et al. (1987).
Results Responses o f c A M P induced by W G A . The t e t r a v a l e n t lectin W G A can i n d u c e all the m a t i n g responses in C. eugametos gametes ( K o o i j m a n et al. 1989). T h a t c A M P
30
Reagents. Phorbol myristate acetate, db-cAMP and db-cGMP were obtained from Sigma, St. Louis, USA. Caffeine was purchased from BDH chemicals, Poole, UK. Calcium ionophore A23187 was purchased from Boehringer, Mannheim, FRG and WGA was obtained from Serva, Heidelberg, FRG. Mating reactions. Twitching was assessed by microscopical observation. Flagellar-tip activation (FTA) was observed and quantitated as described previously (Kooijman et al. 1986). The presence and localization of active agglutinins was assessed with the aid of the monoclonal antibody Mab 66.3 using an indirect immunofluorescence test (Kooijman et al. 1989). Monoclonal antibody Mab 66.3 binds to the binding site of the mt- agglutinin which is exposed on the active but not on the inactive form of the molecule (Homan et al. 1988; Kooijman et al. 1988). Cells were tested for local cell-wall lysis by enclosing a suspension of gametes on a microscope slide under a coverslip. After 5-10 rain, the preparation had dried out to the extent that the cell contents were squeezed out, resulting in the extrusion of a "balloon" between the bases of the flagella (F. Schuring, University of Amsterdam, The Netherlands, personal communication). Mating-structure activation was observed by indirect immunofluorescence assay using WGA at a concentration of 500 pg.ml- 1 or Mab 66.3. Wheat-germ agglutinin and Mab 66.3 bind to the
09 O
,r-
O-
0
i 2
9
i 4
.
,
i
9
9
6
i 8
9
i 10
2
time (min) Fig. l. Agglutination- and WGA-induced changes in the cAMP levels in Chlamydomonas gametes incubated in the light. CyclicAMP concentrations were determined after mixing mt+ and rotgametes (open symbols) or during the incubation of rot- gametes in 500 I.tg-ml-1 WGA (closed symbols). The cAMP levels measured during agglutination are the mean levels in the rot- and the mt+ cells
R. Kooijman et al. : c A M P signal in Chlamydomonas gametes 6O
531 Table 1. Mating responses of Chlamydomonas induced by W G A . Cells that were used in the experiment of Fig. 5 were tested for the induction of mating reactions using W G A in darkness and in light. After 1 min incubation with W G A , the cells were tested for the presence ( - - ) or absence ( - ) of twitching activity; FTA, tipping and cell-wall lysis were tested and quantified after a 30-min incubation period
A
4O 30-
Mating reaction
WGA (gg .ml- 1)
Dark
Light
Twitching Twitching Tipping FTA Cell-wall lysis Cell-wall lysis
25 50 50 50 50 500
-+ 24% 46% 0% 0%
+ + 36% 32% 0% 30%
20-
01
CO
100
9
9
0
'0
I
I
2
4
-6 E
I
9
6
I
8
I0
1
time (min)
d.
